Development and binding mode assessment of N-[4-[2-Propyn-1-yl[(6 S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl] amino]benzoyl]-L-γ-glutamyl-D-glutamic acid (BGC 945), a novel thymidylate synthase inhibitor that targets tumor cells

Article abstract of DOI:10.1021/jm400490e

N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl) -4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-γ-glutamyl-d- glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor glycoprotein, which is overexpressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2′-deoxyuridine-5′-monophosphate, and a model for a similar complex with human TS.

Full text of DOI:10.1021/jm400490e

Article
pubs.acs.org/jmc
Development and Binding Mode Assessment of N‑[4-[2-Propyn-1-
yl[(6S)‑4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-
oxo‑3H‑cyclopenta[g]quinazolin-6-yl]amino]benzoyl]‑L‑γ-
glutamyl‑D‑glutamic Acid (BGC 945), a Novel Thymidylate Synthase
Inhibitor That Targets Tumor Cells
Anna Tochowicz,† Sean Dalziel,‡ Oliv Eidam,§ Joseph D. O’Connell, III,† Sarah Griner,†,⊥
Janet S. Finer-Moore,† and Robert M. Stroud*,†
†Department of Biochemistry and Biophysics, University of CaliforniaSan Francisco, 600 16th Street, San Francisco, California
94158, United States
‡Onyx Pharmaceuticals, Inc., 249 E. Grand Avenue, South San Francisco, California 94080, United States
§Pharmaceutical Chemistry Department, University of CaliforniaSan Francisco, 1700 Fourth Street, Byers Hall, San Francisco,
California 94158, United States
ABSTRACT: N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymeth-
yl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glu-
tamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule
thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer
Research in London. It is licensed by Onyx Pharmaceuticals and is in phase 1
clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed
and raltitrexed that combines enzymatic inhibition of thymidylate synthase
with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential
for efficacy with lower toxicity due to selective intracellular accumulation
through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor
glycoprotein, which is overexpressed mainly in ovarian and lung cancer tumors,
has an affinity for 1 similar to that for its natural ligand, folic acid. This study
describes a novel synthesis of 1, an X-ray crystal structure of its complex with
Escherichia coli TS and 2′-deoxyuridine-5′-monophosphate, and a model for a similar complex with human TS.
synthetase, which normally acts on the cofactor mTHF.
Replacing the N-10 propargyl group and the benzene ring in
CB3717 with a methyl group and thiophene ring, respectively,
significantly improved solubility and potency and decreased the
nephrotoxicity of the compound.13 Importantly, intracellular
polyglutamylation of raltitrexed, which allows its cellular
retention, does not decrease its activity. Raltitrexed is not
approved by US Food and Drug Administration (FDA).
Nevertheless, it became the first new drug for treatment of
colorectal cancer since the mid 1990s, and it was licensed in
Canada and many European countries for the treatment of
metastatic colorectal cancer.16,17 Subsequently, pemetrexed
(LY231514), approved by the FDA in 2004, was licensed for
the treatment of malignant pleural mesothelioma. Pemetrexed
is a multitargeted antifolate,18,19 which in 2008 was granted
approval as a first-line treatment in combination with cisplatin
for treatment of locally advanced and metastatic nonsmall cell
lung cancer. Alone or in combination with other chemo-
therapeutics, pemetrexed also shows activity in a number of
INTRODUCTION
■
Thymidylate synthase (TS) (EC 2.1.1.45) has been recognized
for decades as a key enzyme target for anticancer drugs because
it plays a pivotal role in DNA replication.1−3 TS directly
methylates the C5 of uridine in 2′-deoxyuridine-5′-mono-
phosphate (dUMP), converting it to dTMP, in the sole de
novo synthetic pathway to thymidine, which is required for
DNA replication. Many inhibitors that compete with either the
substrate (dUMP) or the cofactor 5,10-methylene-5,6,7,8-
tetrahydrofolate (mTHF) have been developed as drug leads.
Also many X-ray structures of TS complexes with such
inhibitors have elucidated their mechanisms of inhibition.4−9
The structures encouraged rational design of analogues and
different generations of structure and mechanism-based
antifolate drugs, some of which are in clinical use.7,10 However,
treatment is often complicated by the problems of resistance
and high toxicity.7,11−13
CB371714,15 (Figure 1) is an early quinazoline-based folic
acid analogue. Its clinical development was halted because of
life-threatening renal and hepatic toxicity and poor solubility.13
Raltitrexed (ZD1694) is a slightly modified analogue of
CB3717 that is polyglutamatable by the folylpolyglutamate
Received: April 4, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Chemical structures of the cofactor folic acid and thymidylate synthase inhibitors: CB3717, raltitrexed, pemetrexed, plevitrexed, and 1.
other tumors including head and neck, breast, bladder, cervical,
gastric, pancreatic, ovarian, and colorectal cancers.20−22
Pemetrexed is the first antifolate for which toxicity was reduced
by a low-level folic acid and vitamin B-12 supplement.20,23,24
However, the optimization of folic acid supplementation to the
level that decreases toxicity without compromising the
antitumor effect of the drug still remains difficult and perhaps
must be further explored.25 Plevitrexed (BGC 9331, ZD9331)
is a nonpolyglutamatable inhibitor that was developed as a
result of the raltitrexed and pemetrexed adverse clinical effects.
The effectiveness of plevitrexed demonstrates that polygluta-
mylation is not required for potency of antifolates. It can be
transported to tumor cells via both the α-folate receptor (α-FR)
and the physiological reduced-folate carrier system (RFC).26
Clinical studies evaluating plevitrexed are still ongoing; the
main interest in the drug is as an alternative treatment for
gastric cancers for patients who are not able to tolerate
platinum-based combination therapy.
antifolates. The stereochemistry around the D-amino acid α-C
prevents cleavage by peptide hydrolases. In the α-FR-expressing
human epidermoid (KB) or ovarian (IGROV-1) tumor cells its
IC50 for inhibition of proliferation is ∼1−10 nM and for cells
that do not express the FR it is in low micromolar range.27
Thus, some non-FR-mediated uptake into cells occurs at higher
concentrations. However, short exposure to these higher
concentrations, as observed in the plasma of KB or IGROV-1
tumor-bearing mice dosed with a single bolus injection of 1,
leads to tumor-selective TS inhibition because of rapid
clearance from normal tissues. Compound 1 is licensed by
Onyx Pharmaceuticals and is in phase 1 clinical studies
(ISRCTN 79302332).
We present a synthetic route for 1 and subsequent structural
biology for the 1.7 Å cocrystal structure of the ternary complex
of E. coli TS (ecTS) with dUMP and 1. Further, a model of
human TS (hTS) in complex with dUMP and 1 is reported,
highlighting the impact of chemical modifications on protein
binding.
1 (BGC 945, ONX 0801), Figure 1, was designed to further
reduce toxicity by more effectively targeting cancer cells that
overexpress the α-FR.27 The α-FR is overexpressed in certain
epithelial tumors, particularly ovarian cancer cells (more than
90% overexpress α-FR), and also lung, endometrial, and
mesothelioma tumors.28,29 Importantly, the inhibitor is
selectively transported via the α-FR and has reduced affinity
for the widely expressed bidirectional RFC.27 The RFC is
ubiquitously expressed and responsible for the uptake of
conventional antifolates into normal tissues and hence can
cause TS-related toxicities in the bone marrow and gut. 1
emerged from a lead series of potent inhibitors that displayed
low and high affinity for the RFC and the α-FR respectively
(KD ∼ 1 mM and 1 nM).30 The compound has an L-Glu-γ-D-
Glu moiety that enhances binding to TS, and mimics the
diglutamate metabolites of mTHF and certain conventional
RESULTS
■
Development of Compound 1. The inhibitor 1 (Figure 1
and Scheme 1A) belongs to a series of cyclopenta([g]-
quinazoline-based antifolates for which synthesis and develop-
ment have been described previously.31,32 These compounds
were synthesized by stepwise addition of a p-aminobenzoate
moiety, an N10-substituent, and a dipeptide moiety to a
cyclopenta[g]quinazoline ring. Importantly, an L-Glu-γ-D-Glu
dipeptide moiety replaces the glutamate moiety present in
other antifolates.33,34 Further intracellular polyglutamylation of
TS antifolate inhibitors by folate polyglutamate synthase
increases their affinity for TS.33,34 The L-Glu-γ-D-Glu dipeptide
moiety of 1 serves the same purpose as the polyglutamate tail,
increasing affinity for TS through electrostatic interactions with
B
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 1. (A) Molecular Structure of 1 Trisodium Salt Drug Substance. (B) L-γ-Glutamyl-D-glutamic Acid Tris(1,1-
dimethylethyl) Ester. (C) N-[4-[2-Propynyl-[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-1H-cyclopenta[g]quinazolin-6-
yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic Acid Tris(1,1-dimethylethyl) Ester. (D) N-[4-[2-Propynyl[(6S)-4,6,7,8-tetrahydro-
2-(hydroxymethyl)-4-oxo-1H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic Acid. (E) N-[4-[2-
Propynyl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-1H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-
glutamic Acid Sodium Salt (1:3), ONX 0801 Trisodium Salt Amorphous Drug Substance
C
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the positively charged polyglutamate-binding groove on the
enzyme. The D-Glu protects 1 from peptide hydrolases in the
cell. As mentioned above, several routes of synthesis of 1 are
reported.31,32 For preparation of the sample for X-ray
crystallography as well as the initial phase I clinical study
drug substance, an innovative four-stage synthetic route was
evolved and conducted under Good Manufacturing Practices at
Regis Technologies, Inc. (Morton Grove, IL) (Scheme 1).35,36
Among other improvements, this route did not require the
cobalt catalyst system used in earlier routes.31,32 A molar yield
of 13.3% with respect to the tricyclic acid starting material was
achieved in very good quality. The purity results were 99.5% of
area percent basis (a/a) by achiral HPLC, 99.6% enantiomeric
excess (ee) 6S chiral purity, and 99.8% L,D diastereomeric purity
(see Experimental Section). This process has subsequently
been further improved for larger scale pharmaceutical
production with increased robustness.
Overall Structure of the TS−dUMP−Inhibitor Com-
plex. The structure of the TS ternary complex with the
substrate dUMP and inhibitor 1 elucidates the binding mode of
1 and may be useful in further evolution of this class of drugs.
We attempted to cocrystallize 1 with hTS and substrate,
dUMP, but after extensive screening were only able to obtain
small needles that did not diffract well. We were unable to
optimize these crystal hits, so instead focused on the ecTS
complex. The justification for this approach is that the active
sites of hTS and ecTS are highly conserved and high affinity
hTS inhibitors have been developed based on the high-
resolution crystal structure of an ecTS complex.37
Table 1. Data Collection and Refinement Statistics for 4ISK
data collection
space group
P21
N molecules per AU
cell dimensions (Å), a, b, c (Å), β (deg)
resolution (Å)
8
95.9, 85.5, 134.3 109.4
30.0−1.75
total reflections
unique reflections
completeness (%)
Rmerge (%)
602132
200975
a
99.8 (97.8)
a
7.0 (65.3)
a
redundancy
2.98 (3.04)
a
I/σ(I)
8.79 (1.59)
Wilson B-factor
refinement
20.41
resolution (Å)
29.8−1.75
200689 (4014)
0.18/0.23
37536
35916
368
no. of reflections (test set)
Rwork/Rfree (%)
no. of atoms
protein
ligand (inhibitor 1)
ligand (UMP/UMC)
ions (Mg2+)
245
7
water
1000
average B-factor (Å)
34.40
rms deviations
bond length (Å)
0.013
1.44
bond angels (deg)
a
Statistics for the highest resolution shell are shown in parentheses.
We determined the crystal structure of Escherichia coli TS
(ecTS) in a complex with dUMP and 1 to a resolution of 1.7 Å.
The crystals contained eight independent molecules (four
obligate homodimers) in the asymmetric unit (Table 1). The
second Glu (the D-Glu) of the di-Glu moiety of each inhibitor
protrudes from the protein surface and contributes to the
packing interface with an adjacent pseudo-2-fold related dimer.
A similar interface cannot be formed in hTS because hTS has
inserts with respect to ecTS in the vicinity of the ecTS interface.
In the case of hTS, the di-Glu moiety may actually interfere
with formation of well-ordered crystals, especially if it is
conformationally disordered.
Both protomers contribute to each active site of a dimer. A
molecule of 1 and dUMP are found in each active site. As
expected, the ecTS enzyme is in an active conformation, also
referred to as a closed conformation. This is typical of TS
ternary complexes with substrate and cofactor (or cofactor
analogue) (Figure 2A).8,9,38
defined by the electron density and is schematized in Figure 3
and shown in three dimensions in Figures 4 and 5A. There are
at least three direct polar contacts between the ligand and the
protein observed: the nitrogen at quinazoline position 3 forms a
hydrogen bond with Asp169 (2.6 Å), and the carbonyl oxygen
at position 4 makes a hydrogen bond with Gly173 (3.0 Å). The
2-hydroxymethyl group is within hydrogen bonding distance of
the backbone nitrogen of Ala263 (2.8 Å) and the carboxyl
oxygen of Asp169 (2.9 Å). The nitrogen at position 1 hydrogen
bonds with a conserved structural water molecule (Wat477).
The fused cyclopentyl moiety displaces Trp80, and the
propargyl group is oriented toward Asn177 (Figure 5B). The
benzoate ring of the inhibitor makes a T-shaped edge-to-face
π−π stacking interaction with Phe176.
The L-Glu-γ-D-Glu moiety is a novel feature of 1 whose
binding mode to TS has not been previously characterized. It
binds to ecTS analogously to the polyglutamyl moiety of
polyglutamylated raltitrexed40 except that the D-Glu is rotated
180° about its N-α-C bond. This rotation switches the
orientations of D-Glu’s carboxyl group and side chain compared
to the second Glu moiety (Glu-2) of the raltitrexed
polyglutamate (Figure 4). Thus the carboxyl group lies in the
polyglutamate-binding groove of the protein, whose floor is
formed by His51, Arg53, and Ser54. The major protein contact
with Glu-2 in the raltitrexed complex is a van der Waals
interaction with His51 in which the plane of the His51
imidazole ring is parallel to the plane of the Glu-2-Glu-3 amide
linkage. His51 is in van der Waals interaction with the D-Glu
carboxyl of 1, with the plane of its imidazole ring parallel to the
plane of the D-Glu carboxyl (Figure 4).
Substrate dUMP Binding Mode. The substrate dUMP
and the inhibitor tricyclic ring system are bound in the same
orientation and binding site in each of the subunits in the
asymmetric unit. In five of the subunits there is a covalent bond
between the Sγ of the catalytic Cys146 and C6 of the dUMP
pyrimidine ring. Despite this result, in three of the four dimers
the Cys146 adopts a different rotamer in one of the two active
sites and is not covalently linked to dUMP C6. This asymmetry
in dUMP binding has been seen in other TS ternary complexes
such as the ternary complex of hTS with raltitrexed and dUMP6
and is consistent with the half-of-the-sites reactivity of ecTS.39
A
covalent bond between the catalytic cysteine and the substrate
dUMP is not required for potent TS inhibition by cofactor
analogues.
The ecTS Ternary Complex with dUMP and 1 Has a
Novel Magnesium Cluster. The binding mode of 1 is well-
There are no direct hydrogen bonds between the L-Glu-γ-D-
Glu moiety and the protein, but there are several water-
mediated hydrogen bonds to the protein and two intra-
D
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. (A) Cartoon representation of crystal structure of the ecTS homodimer (blue and gray) with substrate dUMP (green carbons) and
inhibitor 1 (pink carbons) bound at the active site shown in CPK representation. (B) A hydrated magnesium-cluster is found between enzyme and
the second glutamate side chain of inhibitor 1. Final 2Fo − Fc electron density for the inhibitor and Mg2+−water cluster (Mg ion and hydrogen bonds
depicted in green) is shown in blue.
Figure 3. Schematic representation of 1 binding interactions with ecTS generated by the program LigPlot+.41 The covalent bonds of BGC 945 are
depicted in purple, and orange for the protein. Hydrogen bonds are illustrated in green, and hydrophobic interactions are represented as red
eyelashes around the protein residue names and ligand atoms. Only a few waters are shown for clarity (cyan spheres), for example the water
molecules of the magnesium-cluster (Mg2+ in green) near the diglutamate moiety.
2
molecular polar hydrogen bonds (N−H−O−CO) between
the amides and the carboxyl groups. 1 also makes four
hydrogen bonds with water molecules from a newly observed
hexahydrated magnesium ion cluster. The Mg2+ cluster is
sandwiched between the second glutamate side chain
carboxylate of 1 and the side chain of protein residue Glu82,
thereby forming ionic interactions with both the ligand and the
protein (Figure 2B, 3).
Structural Differences between the ecTS Complex
with 1 and dUMP and Its Complex with Raltitrexed and
dUMP. 1 differs from raltitrexed in five regions of the molecule:
(1) the glutamate moiety of raltitrexed is replaced by a L-Glu-γ-
D-Glu moiety; (2) the methyl at C of the quinazoline ring in
raltitrexed is replaced by a hydroxymethyl; (3) in 1, a
cyclopentane group is fused to the quinazoline ring system;
(4) the N10 methyl is replaced by a propargyl group in 1; (5)
the thiophene ring is replaced by a benzene ring (Figure 1).
These molecular differences make 1 (MW 646) a larger
molecule than raltitrexed (MW 458), and the active site has to
expand to accommodate the larger ligand. Thus, superposition
of the structures of the two ecTS ternary complexes reveals
minor changes in the active site residue side chains (Figure 5B),
such as movements of Trp80, Asn177, Leu172, and His51.
However, the backbone atoms of the two complexes closely
E
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
His51. In theory, such an interaction is also possible with Phe80
in hTS, but in the hTS model the carboxylate shifts by 2 Å
toward Arg49. Also, while the diglutamate moiety of 1 makes
exclusively water-mediated hydrogen bonds with ecTS, Lys77
and Lys308 side chains interact directly with 1 in the model of
hTS (Figure 5C). These changes in protein−ligand interactions
are likely observed because the energy minimization was carried
out in the absence of water, but they could also reflect potential
ionic interactions.
DISCUSSION AND CONCLUSIONS
■
Novel drug discovery strategies are pivotal for developing better
cancer therapies. Effectiveness of antifolate drugs used in cancer
treatment is limited by toxicity and mechanisms of drug
resistance, including overexpression of hTS. The thymidylate
synthase inhibitor 1 was developed to reduce toxicity toward
nontumor cells by specifically targeting the α-folate receptor,
which is overexpressed in certain tumor cells. The IC50 for
growth inhibition of α-FR-negative cells by 1 is ∼7 μM
compared to IC50s in the 1−300 nM range for cells
overexpressing the α-FR receptors.27
Figure 4. Stick representation of the inhibitor-binding site in the ecTS
complex focusing on the di-Glu environment. Water-mediated
hydrogen bonds connecting the inhibitor to the protein are shown
as yellow dashes. Waters involved in the network are shown as red
spheres. The inhibitor is shown with pink carbons. The magnesium
cluster is shown with cyan (for water) and green (for Mg2+) spheres.
For comparison, polyglutamylated raltitrexed from an ecTS−dUMP−
raltitrexed complex (PDB 2BBQ) is shown as thin green lines overlaid
on 1.
1 was designed based on experience with a series of
predecessors that bind to TS in a similar manner, primarily
pemetrexed, a multitargeted drug licensed for lung cancer and
mesothelioma treatment, and raltitrexed, which is specific for
colorectal cancer. The novel features of 1 are the cyclopenta-
[g]quinazoline ring system and the side chain L-Glu-γ-D-Glu
moiety. The purpose of this study was to elucidate how these
novel structural features are accommodated in the active site of
hTS to give a potent (Ki ∼ 1.2 nM) inhibitor. The crystal
structure of 1 and dUMP bound to ecTS has been determined
at a resolution of 1.7 Å and used to evaluate the structural
determinants of binding affinity. The high resolution of the
crystal structure allowed accurate description of the inhibitor
binding mode and interactions with the protein.
Two crucial observations emerge from this study. First, the
novel diglutamate moiety binds in a solvent-mediated
association with the surface of TS, analogously to the
polyglutamate moieties of polyglutamylated antifolates. Crystal
structures of ternary complexes of ecTS with polyglutamylated
TS inhibitors have shown that the polyglutamylate tails bind to
an electropositive binding groove on the enzyme surface,
making mainly water-mediated hydrogen bonds with the
protein.7,40 Residues lining the groove are poorly conserved,
which is consistent with the small number of direct hydrogen
bonding interactions. Hence, there is considerable plasticity at
the polyglutamate−protein interface; it is not surprising that
the groove can bind a dipeptide with novel stereochemistry.
The binding site for L-Glu-γ-D-Glu dipeptide, with the D-
enantiomer stabilizing 1 against hydrolysis, overlaps the binding
site for the first two glutamates in the polyglutamate tails, but
with the carboxyl and side-chain groups of the D-Glu in
reversed positions. Thus, the carboxyl of the D-Glu is in the
binding groove while the side chain is oriented away from the
binding groove and toward the protein surface. The D-Glu
makes water-mediated intermolecular hydrogen bonds with
adjacent dimers in the crystal. hTS is not conserved in this
region, and we speculate that if the D-Glu were oriented in a
different conformation, or disordered, these stabilizing crystal
packing interactions could not occur, which may explain our
failure to cocrystallize hTS with 1.
overlap: the rmsd for the α-Cs of the complexes after they are
superposed is 0.4 Å. These mean deviations are similar to the
rmsds between any two complexes in the crystal structure of
the ecTS ternary complex with 1, which has four dimers per
asymmetric unit.
Implication for Binding of 1 to hTS (model). There is
no X-ray structure of 1 bound to hTS available. To understand
how 1 could bind to hTS, the hTS−dUMP−1 complex was
modeled (Figure 5C) based on the structure of ecTS complex
reported here. The suitability of this model is supported by the
similarity of the crystal structures of hTS and ecTS ternary
complexes with dUMP and raltitrexed (rmsd = 0.53 Å over
1500 atoms) (Figure 5E), and the high conservation of the
residues within the folate binding site (12 out of 14 residues in
a 5 Å radius around the ligand are identical, which corresponds
to a sequence identity of 86%).
To accommodate 1 within the hTS active site, hTS residues
within a radius of 5 Å of the ligand were minimized (see
Experimental Section). The binding mode of 1 in the model of
the hTS complex is very similar to the binding mode of 1 in the
crystal structure of the ecTS complex (Figure 5F). The most
profound changes during minimization were observed for
residues Trp109 and Asn226, which rotated by 20° and 30°,
respectively, to accommodate the cyclopentyl and propargyl
groups (Figure 5D). Also, the benzoate ring of 1 displaces L221
and F225. These rearrangements are similar to the structural
differences observed between the ecTS ternary complex with 1
and ecTS−dUMP−raltitrexed (Figure 5B). The hydrogen-
bonding network of the quinazoline ring of 1 is highly
conserved between human and E. coli TS. Subtle differences are
observed in the interactions of the diglutamate moiety. One of
two sequence differences in the binding site of 1 is that His51
in ecTS is Phe80 in hTS. In ecTS, the C-terminal glutamate
carboxylate makes an anion π−π stacking interaction with
As cocrystallization of hTS with 1 was not successful, we
used modeling to investigate how 1 may bind to this enzyme.
F
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Views of ecTS and hTS active sites bound to folate mimetic inhibitors. (A) Crystal structure of ecTS bound to 1 and dUMP. (B) Crystal
structure of ecTS bound to 1 (green carbon atoms) compared to the crystal structure of ecTS bound to raltitrexed (gray carbon atoms, PDB ID
2KCE). (C) Model of hTS bound to 1. A crystallographic water molecule (Wat980) included in the modeling is depicted with hydrogens. (D)
Model of hTS bound to 1 (yellow carbon atoms) compared to the crystal structure of hTS bound to raltitrexed (cyan carbon atoms, PDB ID
1HVY). (E) Superposition of crystal structure of ecTS bound to raltitrexed (gray carbon atoms) on the crystal structure of hTS bound to raltitrexed
(cyan carbon atoms). (F) Superposition of the crystal structure of ecTs bound to 1 (green carbon atoms) on the model of hTS bound to 1 (yellow
carbon atoms).
Human and E. coli TS active sites are highly conserved (86%),
and comparison of crystal structures of ecTS and hTS with
raltitrexed (Figure 5E) showed that the folate inhibitor binding
to both species was almost identical. Thus, similar changes to
those observed in the crystal structure of ecTS−dUMP−1 were
also predicted in a model of hTS−dUMP−1 (Figure 5F).
Analysis of this model revealed that hTS may accommodate the
inhibitor by minor side-chain movements of a few active site
residues, mainly to harbor the cyclopentyl and propargyl
groups. Furthermore, the model predicted two additional direct
polar interactions between the C-terminal glutamate tail and
the human protein (K77 and K308) (Figure 5C). The caveat
with the modeling is that the tail could equally well make water-
mediated contacts with side chains in human TS, but water
molecules were not included in the minimization.
The second key observation is a magnesium ion cluster
linking a loop containing the invariant active site residue Trp80
with the end of the polyglutamate moiety. There is evidence in
the literature that magnesium enhances catalysis by ecTS, but
the basis for this effect was not known.42 Our structure is the
first to show a hexahydrated Mg2+ cluster unambiguously
bound to ecTS. It is consistent with the proposal that Mg2+
binding to a surface groove remote from the active site can
enhance catalysis indirectly, by reducing the entropy of active
site residues.43 The Mg2+ cluster also likely increases affinity of
ecTS for the inhibitor by hydrogen bonding to the D-Glu. The
loop residues that hydrogen bond to the cluster are not
conserved in hTS; thus, a Mg2+ cluster would not likely bind to
the same site in hTS.
The potent and preferential transport of 1 by a receptor that
is overexpressed in cancer cells strongly suggests that 1 might
be the first highly targeted antifolate for cancer treatment. Due
to the well-characterized transport of 1 via α-FR, appropriate
patient selection might benefit from use of diagnostic
techniques to assess the α-FR receptor level prior to initiation
of treatment with this agent.30 This approach would facilitate a
personalized identification of patients most suitable to the
intrinsic cell targeting capability of 1. Our studies have
developed a novel synthetic path to 1 and shown how this
inhibitor binds to the validated cancer drug target TS. The
novel di-Glu moiety on the inhibitor binds in the
polyglutamate-binding groove, and its mode of binding includes
an ionic magnesium cluster coordinated by structural water, the
inhibitor, and multiple residues on the TS enzyme.
G
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
SR-1 Stage 1; L-γ-Glutamyl-D-glutamic Acid Tris(1,1-dimethyleth-
yl) Ester (Scheme 1B). N-[N-[(Phenylmethoxy)carbonyl]-L-γ-glutam-
yl]-D-glutamic acid, tris(1,1-dimethylethyl) ester, 3.5 kg, was dissolved
in ethanol and hydrogenated in the presence of 5% palladium on
carbon under a 20 pound per square inch (gauge) hydrogen
atmosphere. After the uptake of hydrogen ceased, the reaction was
filtered through Celite to remove the catalyst, and the catalyst cake was
washed with methylene chloride. The resulting solution was stripped
to dryness, dissolved in ethylene dichloride, and then stripped to
dryness under reduced pressure to give 2.54 kg (approximately 94%
yield) of stage 1 product as a clear yellow oil. Ethylene dichloride was
removed through subsequent processing in the remainder of the
synthesis and verified by gas chromatography of the drug substance to
be below <5 ppm.
SR-1 Stage 2; N-[4-[2-Propynyl-[(6S)-4,6,7,8-tetrahydro-2-(hy-
droxymethyl)-4-oxo-1H cyclopenta[g]quinazolin-6-yl]amino]-
benzoyl]-L-γ-glutamyl-D-glutamic Acid Tris(1,1-dimethylethyl) Ester
(Scheme 1C). 4-[2-Propyn-1-yl-[6S-4,6,7,8,-tetrahydro-2-(hydroxyl-
methyl)-4-oxo-3H-cyclopenta[g]quinazolin-yl]amino]benzoic acid,
1.52 kg, was charged to 32 kg of dimethylformamide (DMF) solvent.
To this mixture was added a solution of 2.53 kg L-γ-glutamyl]-D-
glutamic acid tris(1,1-dimethylethyl) ester (stage 1) dissolved in 10.1
kg of DMF. To this combined mixture were added 0.79 kg of 1-
hydroxybenzotriazole hydrate, 0.87 kg of N-methylmopholine, and
0.82 kg of dimethylaminopropyl ethylcarbodiimide hydrochloride.
This mixture was stirred at room temperature for 12 h, and then
poured into water and extracted into ethyl acetate. The product
containing ethyl acetate portion was stripped to dryness to obtain 3.13
kg of oil. This material was purified using silica gel chromatography,
eluting with methanol/methylene chloride. The eluent was stripped to
dryness to give 1.22 kg (38% yield) of the stage 2 product.
SR-1 Stage 3; N-[4-[2-Propynyl[(6S)-4,6,7,8-tetrahydro-2-(hydrox-
ymethyl)-4-oxo-1H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-
γ-glutamyl-D-glutamic Acid (Scheme 1D). N-[4-[2-Propynyl[(6S)-
4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-1H-cyclopenta[g]-
quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid tris(1,1-
dimethylethyl) ester (stage 2), 407 g, was dissolved in 4.1 L of
trifluoroacetic acid and stirred at 0 °C to 5 °C for about 2 h. The
triflouroacetic acid was removed by vacuum distillation and replaced
with 17 L of methylene chloride. The methylene chloride was removed
by vacuum distillation, and a red oil resulted. Methyl tert-butyl ether
(MTBE, 3.4 L) was charged to the oil, and the mixture was stirred
until a solid resulted. The solid was isolated by filtration, rinsed on the
filter with MTBE, slurried in acetonitrile, filtered, and dried to give 292
g (77% yield) of stage 3 product.
SR-1 Stage 4; N-[4-[2-Propynyl[(6S)-4,6,7,8-tetrahydro-2-(hydrox-
ymethyl)-4-oxo-1H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-
γ-glutamyl-D-glutamic Acid Sodium Salt (1:3), ONX 0801 Trisodium
Salt Amorphous Drug Substance Synthesis (Scheme 1E). The stage
3 product, 557 g, was slowly added to a solution of 278 g of sodium
bicarbonate and 5.5 L of water, resulting in a clear solution. This
solution was added to a column containing 32 kg of Sepabeads SP-
207SS (Resindion, Mitsubishi Chemical Corporation) and eluted with
water, followed by dilute acetonitrile in water. The pure product-
containing fractions were combined, filtered through a 2 μm cartridge,
and lyophilized. The lyophilized solid was redissolved in 20 L of water
and ultrafiltered using a 5000 MW cutoff cartridge. The filtrate was
lyophilized to give 370 g (67% yield) of a light-beige powder, ONX
0801 trisodium salt amorphous drug substance with a purity by HPLC
of 99.5% a/a, by achiral HPLC 99.6% ee 6S chiral purity, and 99.8%
L,D diastereomeric purity.
EXPERIMENTAL SECTION
■
1. Cells, Cell Culture, Protein Expression, and Purification.
ecTS was expressed in DH5α cells at 37 °C, induced at OD600 0.6−0.8
with 1 mM IPTG for 4 h. Enzyme was purified by ion exchange
chromatography (Q column) as described by Boles et al.44 and
subsequently dialyzed into 20 mM KPO4 pH 7.4, 100 mM NaCl, and 2
mM DTT. The purification was then confirmed by SDS-PAGE gel.
2. Protein Crystallization. An N-terminally His-tagged construct
that was previously cocrystallized with inhibitors45 was used for
screening crystallization conditions for the ternary complex of hTS
with dUMP and 1. Several published protocols for cocrystallizing hTS
with dUMP and antifolate inhibitors were tried, and small crystals were
obtained using the protocol used by Almog et al.6 However, these
diffracted to less than 5 Å resolution or were twinned. Some crystal
hits were also obtained from commercial crystallization screens, but
these could not be optimized. Therefore, 1 was cocrystallized with
ecTS and dUMP.
ecTS was concentrated to around 6 mg/mL, dUMP was added to a
final concentration of 3.3 mM, and the inhibitor 1 was added to a final
concentration of 3.3 mM. Additional inhibitor was added right before
the crystal setup. The optimized final crystallization condition were
20−21% PEG 8000, 0.1 M TRIS pH 7.9, 0.2 M MgCl2, and 5 mM
DTT at room temperature. To obtain better crystals, conditions were
optimized using additive screens. Finally, much better diffraction with
MnCl2 and ethanol (added separately) was observed, and data were
collected. Crystals were grown in hanging drops by the vapor diffusion
method using a Mosquito nanoliter-scale robotic workstation (TTP
Labtech). Crystals reached final size in ∼15 days.
3. X-ray Data Collection and Structure Determination. X-ray
diffraction data were collected from cryoprotected crystals (in 20%
ethylene glycol) at Beamline 8.3.1 of the Advanced Light Source
(Lawrence Berkeley National Laboratories) on a 315r CCD detector.
The monoclinic crystals diffracted to 1.75 Å resolution. Data were
indexed, reduced, and scaled with XDS.46 The structure was
determined by molecular replacement using Phaser47 and using
liganded ecTS with the ligands removed (1AXW) as a search model.
Restrained positional refinement and refinement of isotropic B-factors
and TLS refinement were carried out using Phenix.48 NCS restraints
were not used because of small but significant conformational
differences between the subunits in the asymmetric unit. Manual
building was done using Coot.49 Data collection, processing statistics,
R factors, and statistics for the final structure are listed in Table 1.
4. Modeling of the hTS Complex with dUMP and 1. Inhibitor
1 was modeled in the structure of hTS using the program PLOP
(Protein Local Optimization Program).50 1 and dUMP were prepared
using Schrodinger’s hetgrp_ffgen utility bundled with PRIME.51 The
structure of hTS bound to raltitrexed (PDB ID: 1HVY)38 served as a
template for the hTS model. One conserved water molecule (Wat980)
from 1HVY was included in the minimization. 1 was placed in the hTS
active site by superposing the ecTS−dUMP−1 complex (chain A) on
hTS (1HVY, chain A). Protein residues within a 5 Å radius of 1 were
minimized to remove clashes of 1 with hTS. After the protein
minimization, 1 was minimized in the enlarged binding site. dUMP
was modeled without a covalent link to Cys195, as was observed in
several subunits of the ecTS−dUMP−1 structure.
5. Synthetic Chemistry and Development of 1. Inhibitor 1 is
D-glutamic acid, N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hy-
droxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]-
benzoyl]-L-γ-glutamyl-, sodium salt (1:3); CAS registry number
1097638-00-0. The chemical structure of 1 is shown in Figure 1 and
Scheme 1A. The molecular formula of the trisodium salt is
C32H30N5Na3O10 and has a molecular weight of 713.58 g/mol. The
solid form is amorphous and hygroscopic and with a glass transition
temperature of approximately 120.6 °C.
Synthetic Chemistry. General Information. A synthesis of 1 drug
substance was established via a four-stage process referred to as the
SR-1 process, and performed under contract by Regis Technologies
(Morton Grove, IL). Chemicals and reagents were purchased from
commercial suppliers and were used without further purification.
ASSOCIATED CONTENT
■
Accession Codes
Atomic coordinates have been deposited in the Protein Data
Bank as entry 4ISK.
H
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(10) Jackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown,
M.; Calvert, A. H.; Judson, I. R.; Hughes, L. R. ICI D1694, a
quinazoline antifolate thymidylate synthase inhibitor that is a potent
inhibitor of L1210 tumor cell growth in vitro and in vivo: A new agent
for clinical study. Cancer Res. 1991, 51, 5579−5586.
(11) Farrugia, D. C.; Ford, H. E.; Cunningham, D.; Danenberg, K.
D.; Danenberg, P. V.; Brabender, J.; McVicar, A. D.; Aherne, G. W.;
Hardcastle, A.; McCarthy, K.; Jackman, A. L. Thymidylate synthase
expression in advanced colorectal cancer predicts for response to
raltitrexed. Clin. Cancer Res. 2003, 9, 792−801.
(12) Jarmula, A. Antifolate inhibitors of thymidylate synthase as
anticancer drugs. Mini Rev. Med. Chem. 2010, 10, 1211−1222.
(13) Jackman, A. L.; Forster, M.; Ng, M. Targeting thymidylate
synthase by antifolate drugs for the treatment of cancer. In Cancer
Drug Design and Discovery; Neidle, S., Ed.; Elsevier: New York, 2007;
pp 198−226.
(14) Jones, T. R.; Calvert, A. H.; Jackman, A. L.; Brown, S. J.; Jones,
M.; Harrap, K. R. A potent antitumour quinazoline inhibitor of
thymidylate synthetase: synthesis, biological properties and therapeutic
results in mice. Eur. J. Cancer 1981, 17, 11−19.
(15) Clarke, S. J.; Jackman, A. L.; Judson, I. R. The history of the
development and clinical use of CB 3717 and ICI D1694. Adv. Exp.
Med. Biol. 1993, 339, 277−287, Discussion. 289−290.
(16) Wilson, K. S.; Fitzgerald, C. A.; Barnett, J. B.; Gill, S.; Khoo, K.
E. Adjuvant therapy with raltitrexed in patients with colorectal cancer
intolerant of 5-fluorouracil: British Columbia Cancer Agency
experience. Cancer Invest. 2007, 25, 711−714.
(17) Wilson, K. S.; Malfair Taylor, S. C. Raltitrexed: optimism and
reality. Expert Opin. Drug Metab. Toxicol. 2009, 5, 1447−1454.
(18) Walling, J. From methotrexate to pemetrexed and beyond. A
review of the pharmacodynamic and clinical properties of antifolates.
Invest. New Drugs 2006, 24, 37−77.
(19) Goldman, I. D.; Zhao, R. Molecular, biochemical, and cellular
pharmacology of pemetrexed. Semin. Oncol. 2002, 29, 3−17.
(20) Adjei, A. A. Pemetrexed (ALIMTA), a novel multitargeted
antineoplastic agent. Clin. Cancer Res. 2004, 10, 4276s−4280s.
(21) Adjei, A. A. Clinical studies of pemetrexed and gemcitabine
combinations. Ann. Oncol. 2006, 17 (Suppl 5), v29−32.
(22) Tomao, F.; Panici, P. B.; Frati, L.; Tomao, S. Emerging role of
pemetrexed in ovarian cancer. Expert Rev. Anticancer Ther. 2009, 9,
1727−1735.
(23) Scagliotti, G. V.; Shin, D. M.; Kindler, H. L.; Vasconcelles, M. J.;
Keppler, U.; Manegold, C.; Burris, H.; Gatzemeier, U.; Blatter, J.;
Symanowski, J. T.; Rusthoven, J. J. Phase II study of pemetrexed with
and without folic acid and vitamin B12 as front-line therapy in
malignant pleural mesothelioma. J. Clin. Oncol. 2003, 21, 1556−1561.
(24) Vogelzang, N. J.; Rusthoven, J. J.; Symanowski, J.; Denham, C.;
Kaukel, E.; Ruffie, P.; Gatzemeier, U.; Boyer, M.; Emri, S.; Manegold,
C.; Niyikiza, C.; Paoletti, P. Phase III study of pemetrexed in
combination with cisplatin versus cisplatin alone in patients with
malignant pleural mesothelioma. J. Clin. Oncol. 2003, 21, 2636−2644.
(25) Chattopadhyay, S.; Moran, R. G.; Goldman, I. D. Pemetrexed:
biochemical and cellular pharmacology, mechanisms, and clinical
applications. Mol. Cancer Ther. 2007, 6, 404−417.
AUTHOR INFORMATION
Corresponding Author
*E-mail: stroud@msg.ucsf.edu. Phone: 415-476-4224. Fax:
415-476-1902.
■
Present Address
⊥Department of Chemistry and Biochemistry, University of
California, Los Angeles, 611 Charles Young Dr. East, Los
Angeles, CA 90095-1569.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research was supported by NIH grant GM 051232. We thank
Dr. James Holton and Dr. George Meigs at the Advanced Light
Source in Berkeley, at beamline 8.3.1. for their assistance during
data collection.
ABBREVIATIONS USED
■
TS, thymidylate synthase; hTS, human thymidylate synthase;
ecTS, Escherichia coli thymidylate synthase; dUMP, 2′-
deoxyuridine-5′-monophosphate; TMP, thymidine-5′-mono-
phosphate; mTHF, N5,N10-methylene-5,6,7,8-tetrahydrofo-
late; α-FR, α-folate receptor; RFC, reduced-folate carrier
system; DDT, dichlorodiphenyltrichloroethane; PMSF, phenyl-
methylsulfonyl fluoride; a/a, area percent basis; ee, enantio-
meric excess
REFERENCES
■
(1) Heidelberger, C.; Danenberg, P. V.; Moran, R. G. Fluorinated
pyrimidines and their nucleosides. Adv. Enzymol. Relat. Areas Mol. Biol.
1983, 54, 58−119.
(2) Santi, D. V.; Danenberg, P. V. Folates in Pyrimidine Nucleotide
Biosynthesis. In Folates and Pterins; Blakley, R. L.; Benkovik, S. J., Eds.;
John Wiley and Sons: New York, 1984; Vol. 1, pp 345−398.
(3) Jackman, A. L.; Newell, D. R.; Gibson, W.; Jodrell, D. I.; Taylor,
G. A.; Bishop, J. A.; Hughes, L. R.; Calvert, A. H. The biochemical
pharmacology of the thymidylate synthase inhibitor, 2-desamino-2-
methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583). Biochem.
Pharmacol. 1991, 42, 1885−1895.
(4) Stout, T. J.; Stroud, R. M. The complex of the anti-cancer
therapeutic, BW1843U89, with thymidylate synthase at 2.0 A
resolution: implications for a new mode of inhibition. Structure
1996, 4, 67−77.
(5) Stout, T. J.; Tondi, D.; Rinaldi, M.; Barlocco, D.; Pecorari, P.;
Santi, D. V.; Kuntz, I. D.; Stroud, R. M.; Shoichet, B. K.; Costi, M. P.
Structure-based design of inhibitors specific for bacterial thymidylate
synthase. Biochemistry 1999, 38, 1607−1617.
(6) Almog, R.; Waddling, C. A.; Maley, F.; Maley, G. F.; Van Roey, P.
Crystal structure of a deletion mutant of human thymidylate synthase
Delta (7−29) and its ternary complex with Tomudex and dUMP.
Protein Sci. 2001, 10, 988−996.
(7) Sayre, P. H.; Finer-Moore, J. S.; Fritz, T. A.; Biermann, D.; Gates,
S. B.; MacKellar, W. C.; Patel, V. F.; Stroud, R. M. Multi-targeted
antifolates aimed at avoiding drug resistance form covalent closed
inhibitory complexes with human and Escherichia coli thymidylate
synthases. J. Mol. Biol. 2001, 313, 813−829.
(8) Montfort, W. R.; Perry, K. M.; Fauman, E. B.; Finer-Moore, J. S.;
Maley, G. F.; Hardy, L.; Maley, F.; Stroud, R. M. Structure, multiple
site binding, and segmental accommodation in thymidylate synthase
on binding dUMP and an anti-folate. Biochemistry 1990, 29, 6964−
6977.
(9) Matthews, D. A.; Appelt, K.; Oatley, S. J.; Xuong, N. H. Crystal
structure of Escherichia coli thymidylate synthase containing bound 5-
fluoro-2′-deoxyuridylate and 10-propargyl-5,8-dideazafolate. J. Mol.
Biol. 1990, 214, 923−936.
(26) Theti, D. S.; Jackman, A. L. The role of alpha-folate receptor-
mediated transport in the antitumor activity of antifolate drugs. Clin.
Cancer Res. 2004, 10, 1080−1089.
(27) Gibbs, D. D.; Theti, D. S.; Wood, N.; Green, M.; Raynaud, F.;
Valenti, M.; Forster, M. D.; Mitchell, F.; Bavetsias, V.; Henderson, E.;
Jackman, A. L. BGC 945, a novel tumor-selective thymidylate synthase
inhibitor targeted to alpha-folate receptor-overexpressing tumors.
Cancer Res. 2005, 65, 11721−11728.
(28) Jackman, A. L.; Theti, D. S.; Gibbs, D. D. Antifolates targeted
specifically to the folate receptor. Adv Drug Delivery Rev. 2004, 56,
1111−1125.
(29) Elnakat, H.; Ratnam, M. Distribution, functionality and gene
regulation of folate receptor isoforms: implications in targeted therapy.
Adv. Drug Delivery Rev. 2004, 56, 1067−1084.
I
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(30) Pillai, R. G.; Forster, M.; Perumal, M.; Mitchell, F.; Leyton, J.;
Aibgirhio, F. I.; Golovko, O.; Jackman, A. L.; Aboagye, E. O. Imaging
pharmacodynamics of the alpha-folate receptor-targeted thymidylate
synthase inhibitor BGC 945. Cancer Res. 2008, 68, 3827−3834.
(31) Henderson, E. A.; Bavetsias, V.; Theti, D. S.; Wilson, S. C.;
Clauss, R.; Jackman, A. L. Targeting the alpha-folate receptor with
cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase.
Bioorg. Med. Chem. 2006, 14, 5020−5042.
(32) Bavetsias, V.; Marriott, J. H.; Melin, C.; Kimbell, R.; Matusiak,
Z. S.; Boyle, F. T.; Jackman, A. L. Design and synthesis of
cyclopenta[g]quinazoline-based antifolates as inhibitors of thymidylate
synthase and potential antitumor agents. J. Med. Chem. 2000, 43,
1910−1926.
(47) Storoni, L. C.; McCoy, A. J.; Read, R. J. Likelihood-enhanced
fast rotation functions. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
60, 432−438.
(48) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger, T.
R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.;
Sauter, N. K.; Terwilliger, T. C. PHENIX: building new software for
automated crystallographic structure determination. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 2002, 58, 1948−1954.
(49) Emsley, P.; Cowtan, K. Coot: model-building tools for
molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
60, 2126−2132.
(50) Kalyanaraman, C.; Bernacki, K.; Jacobson, M. P. Virtual
screening against highly charged active sites: identifying substrates of
alpha−beta barrel enzymes. Biochemistry 2005, 44, 2059−2071.
(51) Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R.
Novel procedure for modeling ligand/receptor induced fit effects. J.
Med. Chem. 2006, 49, 534−553.
(33) Bavetsias, V.; Jackman, A. L.; Kimbell, R.; Gibson, W.; Boyle, F.
T.; Bisset, G. M. Quinazoline antifolate thymidylate synthase
inhibitors: gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-
desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).
J. Med. Chem. 1996, 39, 73−85.
(34) Jodrell, D. I.; Gibson, W.; Bisset, G. M.; Boyle, F. T.; Judson, I.
R.; Jackman, A. L. The in vivo metabolic stability of dipeptide
analogues of the quinazoline antifolate, ICI 198583, in mice. Biochem.
Pharmacol. 1993, 46, 2229−2234.
(35) Onyx Pharmaceuticals. Efficient peptide couplings and their use in
the synthesis and isolation of a cyclopenta[g]quinazoline trisodium salt.
Application No. PCT/US2011/065752, Publication No. WO 2012/
087888 A2, January 28, 2012.
(36) Onyx Pharmaceuticals. Synthesis of cyclopentaquinazolines.
Application No. PCT/US2011/001235, Publication No. WO 2012/
011939 A2, January 26, 2012.
(37) Reich, S. H.; Fuhry, M. A.; Nguyen, D.; Pino, M. J.; Welsh, K.
M.; Webber, S.; Janson, C. A.; Jordan, S. R.; Matthews, D. A.; Smith,
W. W.; et al. Design and synthesis of novel 6,7-imidazotetrahy-
droquinoline inhibitors of thymidylate synthase using iterative protein
crystal structure analysis. J. Med. Chem. 1992, 35, 847−858.
(38) Phan, J.; Koli, S.; Minor, W.; Dunlap, R. B.; Berger, S. H.;
Lebioda, L. Human thymidylate synthase is in the closed conformation
when complexed with dUMP and raltitrexed, an antifolate drug.
Biochemistry 2001, 40, 1897−1902.
(39) Spencer, H. T.; Villafranca, J. E.; Appleman, J. R. Kinetic scheme
for thymidylate synthase from Escherichia coli: determination from
measurements of ligand binding, primary and secondary isotope
effects, and pre-steady-state catalysis. Biochemistry 1997, 36, 4212−
4222.
(40) Kamb, A.; Finer-Moore, J.; Calvert, A. H.; Stroud, R. M.
Structural basis for recognition of polyglutamyl folates by thymidylate
synthase. Biochemistry 1992, 31, 9883−9890.
(41) Laskowski, R. A.; Swindells, M. B. LigPlot+: multiple ligand−
protein interaction diagrams for drug discovery. J Chem. Inf. Model.
2011, 51, 2778−2786.
(42) Maley, G. F.; Maley, F.; Baugh, C. M. Differential inhibition of
host and viral thymidylate synthetases by folylpolyglutamates. J. Biol.
Chem. 1979, 254, 7485−7487.
(43) Wang, Z.; Sapienza, P. J.; Abeysinghe, T.; Luzum, C.; Lee, A. L.;
Finer-Moore, J. S.; Stroud, R. M.; Kohen, A. Mg2+ binds to the surface
of thymidylate synthase and affects hydride transfer at the interior
active site. J. Am. Chem. Soc. 2013, 135, 7583−7592.
(44) Boles, J. O.; Cisneros, R. J.; Weir, M. S.; Odom, J. D.;
Villafranca, J. E.; Dunlap, R. B. Purification and characterization of
selenomethionyl thymidylate synthase from Escherichia coli: Compar-
ison with the wild-type enzyme. Biochemistry 1991, 30, 11073−11080.
(45) Carosati, E.; Tochowicz, A.; Marverti, G.; Guaitoli, G.;
Benedetti, P.; Ferrari, S.; Stroud, R. M.; Finer-Moore, J.; Luciani, R.;
Farina, D.; Cruciani, G.; Costi, M. P. Inhibitor of ovarian cancer cells
growth by virtual screening: A new thiazole derivative targeting human
thymidylate synthase. J. Med. Chem. 2012, 55, 10272−10276.
(46) Kabsch, W. Integration, scaling, space-group assignment and
post-refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66,
133−144.
J
dx.doi.org/10.1021/jm400490e | J. Med. Chem. XXXX, XXX, XXX−XXX